1. Field of the Invention
The present invention relates to a novel gene of HIV, the virus causing AIDS, which encodes a protein having an amino acid sequence that is closely related to the chemokine family of proteins. More particularly, the invention relates to a novel HIV protein that may be a cofactor for binding to the chemokine receptor on human cells during the entry phase of infection.
2. Description of the Background and Related Art
1. The Chemokine Receptors as Coreceptor for HIV Infection
Efficient entry of HIV into target cells is dependent upon a high affinity binding of the viral envelope glycoprotein, gp120, to the amino terminal domain of CD4, a protein expressed on the surface of the target cell. While CD4 is the primary virus receptor, CD4 alone is not sufficient for virus entry. Chemokine receptors have been identified as the coreceptors involved in the entry of HIV into target cells.
Macrophage-tropic (xe2x80x9cM-tropicxe2x80x9d) HIV-1 use the xcex2-chemokine receptor CCR5, and less often receptor CCR3, as their coreceptor (Choe et al., 1996, Cell 85:1135-1148; Dragic et al., 1996, Nature 381:667-673; Deng et al., 1996, Nature 381:661-666). Mutations in CCR5 appears to confer resistance to infection by M-tropic HIV-1 viruses in vivo and in vitro (Samson et al., 1996, Nature 382:722-725). T-tropic (lymphotropic strains which grow in cells including transformed T cell lines) HIV generally use the xcex1-chemokine receptor CXCR4 (also known as fusin, SDF-1 chemokine receptor, LESTR; Feng et al., 1996, Science 272:872-877). CXCR4 also can function as the primary receptor for HIV-2 entry and infection of human CD4-negative cells (Endres et al., 1996, Cell 87:745-756). Dual-tropic primary HIV-1 isolates, that can infect both macrophages and T cells, can use either CCR5 or CXCR4 (and possibly CCR3 or CCR-2b) as the coreceptor involved in virus entry (Doranz et al., 1996, Cell 85:1149-1158). There is evidence suggesting that the structure of the gp120 V3 loop influences the ability of HIV to bind the chemokine receptors on the target cell (Choe et al., 1996, supra; Doranz et al., 1996, supra).
2. HIV Secondary Structures
Single stranded RNA form localized regions of secondary structures such as hairpin loops and pseudoknot structures (Schimm, 1989, Cell, 58-9-12). A RNA population was isolated that bound to HIV reverse transcriptase and that has a pseudoknot consensus (Tuerk et al., 1992, Proc. Natl. Acad. Sci., USA. 59:6988-6992). Pseudoknots are structures in which there is an intramolecular base pairing of the xe2x80x9cloopxe2x80x9d sequence of an RNA hairpin to sequences either 5xe2x80x2 or 3xe2x80x2 to that hairpin. Pseudoknots are generally formed in nucleic acid sequences of about 30 to 60 nucleotides. Such intramolecular base pairing is key to the translation of RNA since the presence of pseudoknots can lead to frameshifting either in the 5xe2x80x2 or the 3xe2x80x2 direction (generally designated as xe2x88x921 or +1) or for allowing read-through. Translational frameshifting allows the expression of alternative translational products in a predictable stoichiometry (ala retroviral or HIV gag-pol fusion peptide); to allow the expression of alternative enzymatic activities; or as a mechanisms for autogenous control (see Farabaugh, 1996, Microbiol Rev. 104).
3. Chemokines
Chemokines are a superfamily of soluble proteins that are involved in immune regulation and in inflammatory processes (such as leukocyte recruitment). Generally, chemokines range in size from about 70 to about 100 amino acids, and in molecular size from about 8 kilodaltons (kD) to about 11 kD. Chemokine like proteins have also been described that are membrane bound (Pan et a., 1997, Nature, 387:611). The chemokines share related primary structure, particularly with a conserved motif of four cysteine residues. Early classification of chemokines was based on whether the first two cysteines are adjacent to each other (xe2x80x9cCC chemokinesxe2x80x9d), or are separated by one amino acid (xe2x80x9cCXC chemokinesxe2x80x9d). More recently, chemokines with a single xe2x80x9cCxe2x80x9d motif (for example lymphotactin) and xe2x80x9cCXXXCxe2x80x9d motif (for example, neutotactin) have been described. The xcex1-chemokine receptor CXCR4 has been identified as a coreceptor required for HIV entry. The only known natural ligand for CXCR4 has been identified as the CXC chemokine SDF-1. SDF-1 has been shown to inhibit infection of CXCR4 and CD4 expressing cells by T-tropic HIV-1 strains (Oberlin et al., 1996, Nature 382:833-835). Thus, modified versions of chemokines are being tested to determine whether they may be used to block chemokine receptors from binding by HIV.
Kaposi""s sarcoma is an AIDS-related malignancy. The Kaposi""s sarcoma-associated herpesvirus (KHSV, human herpesvirus 8) has been shown to encode a chemokine receptor (xe2x80x9cGPCRxe2x80x9d) that is analogous in sequence and chemokine specificity to CXCR2 (Arvantikas et al., 1997, Nature 385:347-349). This is not the only instance in which a virus has apparently pirated a cellular gene encoding either a chemokine or a chemokine receptor. KSHV and Molluscum contagiosum have open reading frames that encode CC chemokines; and Herpesvirus Saimiri, human cytomegalovirus, KSHV, Equine herpesvirus-2, Swine pox virus, and capripox virus have open reading frames encoding chemokine receptors (Murphy, 1997, Nature 385:296-299; Neote et al., 1993, Cell 72:415-425).
4. HIV Proteins
The HIV genome is known to contain 8 open reading frames on the minus strand of the double-stranded DNA intermediate. From the HIV double-stranded intermediate, and from the HIV promoter located in the 5xe2x80x2 LTR, mRNAs of plus strand polarity are transcribed from the minus strand DNA template (see Definitions section herein). Depending on the processing of the transcript, the mRNA may then be translated into one or more viral proteins including Gag, Pol, Vif, Tat, Vpu, Vpr, Rev, Env, and Nef. Additionally, ribosomal frameshifting is employed to enable gag pol protein. Effective transcription from the 5xe2x80x2LTR HIV promoter is dependent on the presence of Tat for transcriptional activation that dramatically increases the levels of viral mRNAs. A possibility was raised that the plus strand of the viral DNA contains a long open reading frame (ORF), located in the region of the genom complementary to the env gene sequence, that may encode a viral protein of 190 amino acids and a molecular mass of 20 kilodaltons (Miller, 1988, Science 239:1420-1422). However, it is not apparent whether this possibility was confirmed, such as by the demonstration of the putative protein or its respective mRNA. In fact, it is noted in the publication that it is possible that the ORF does not represent a true gene sequence. The possibility that bidirectional transcription occurs in HIV was further evaluated by Michael et al. (1994, J. Virol. 979-87).
Accordingly, there has been and continues to be a long-felt need for the identification of novel HIV proteins which play a role in AIDS pathogenesis, and thus may be important targets of therapeutic intervention.
The present invention relates to the discovery of a novel gene comprising a open reading frame (ORF) in the plus strand of the viral DNA, and located in the HIV LTR. An antisense initiator element initiates production by RNA polymerase of RNA transcripts of negative strand polarity (antisense RNA) utilizing the plus strand DNA as a template. Thus, using this mechanism, the novel HIV gene is transcribed by the cellular transcriptional apparatus. The gene encodes a protein that is related to, and has a structural motif resembling that of a chemokine. More particularly, the protein has similarity to the chemokine family of proteins. These objects and further features and advantages of the invention will be better understood from the description of the preferred embodiments when considered in relation to the figures.